Open Access Repository of Indian Theses

Bellamkonda, Latha

 Abstract 
 i 
THIOPSEUDOUREA LIGATED PALLADIUM(II) COMPLEXES: 
SYNTHESIS, CHARACTERIZATION AND APPLICATION AS 
CATALYSTS FOR C-C COUPLING AND 
HETEROANNULATION REACTIONS 
 
The thesis deals with the design and development of a new class of 
phosphine-free palladium catalysts for C–C coupling and 
heteroannulation reactions. The first chapter contains two sections. 
Section-A deals with the importance of palladium catalysis and major 
problems encountered in the conventional homogeneous process. The 
need for design and development of phosphine-free Pd catalysts for 
higher order of activity is also discussed in this chapter. Section-B deals 
with the preparation and characterization of thiopseudourea 
palladium(II) complexes. Chapter 2 deals with the catalytic activity of 
thiopseudourea palladium(II) complexes in the Suzuki and Hiyama 
reactions of aryl halides. Chapter 3 deals with the thiopseudourea 
palladium(II) complexes catalyzed Heck and Sonogashira reactions of aryl 
halides. Chapter 4 deals with the synthesis of 2,3-disubstituted indoles 
by heteroannulation using thiopseudourea Pd(II) complexes as high 
turnover catalysts for the aryl iodides.  
CHAPTER-I:  
Section-A: Introduction 
This section introduces the-state-of-the-art practiced in industry and 
laboratory in the area of palladium catalysis both in homogeneous and 
heterogeneous conditions. We highlight the importance of ligands for 
 Abstract 
 ii 
homogeneous catalysis and the necessity to design and develop the 
phosphine-free ligands for palladium catalysis. The properties and 
structures of the ligands like phosphine and phosphine-free (N-
heterocyclic carbenes, carbocyclic carbenes) N-donor ligands are 
discussed to familiarize the ligands used for the development of 
homogeneous catalysts.  
 Section-B: Design, Synthesis, and Characterization of 
Thiopseudourea Ligands and Their Pd(II) Complexes  
This section describes the synthesis, characterization of phosphine-free 
N,N,O-tridentate thiopseudourea ligands and their corresponding Pd(II) 
complexes. A brief description has been given below. 
In contrast to phosphine ligands, the N-donor ligands are stable, 
inexpensive, easily synthesizable and less toxic. Accordingly, it is highly 
desirable to develop phosphine-free homogeneous Pd-catalysts for 
palladium catalyzed coupling reactions. New homogeneous palladium 
catalysts based on the simple N,N,O-donor ligands were designed and 
developed (Scheme 1 and 2). The anionic amide (deprotonated amide) 
ligands were chosen which is expected not only to stabilize the 
catalytically active palladium particles but also to provide the adequate 
electron density to the Pd(0) species to facilitate oxidative addition.  
 
 
 
 Abstract 
 iii 
 
 
Cl
O
1. NH4SCN
Acetone, 0 °C
2.Picolyl amine
N
H
N
H
N
O S
Benzyl bromide,
                NaH THF,  0 °C
N
H
N
N
O S
R1
R2
R2
R1
R2
R2
R1
R2
R2
2a-f
1a-f
      R1        R2
a = H        H
b = F        H
c = Br       H
d = I          H
e = CH3    H
f = OCH3  OCH3
 
Scheme 1. Synthesis of thiopseudourea ligands. 
 
N
H
N
N
O S
R1
R2
R2
2a-f
      R1        R2
a = H        H
b = F        H
c = Br       H
d = I         H
e = CH3    H
f = OCH3  OCH3
N
O
Pd
N
S
NR2
R1
R2
OAc
Pd(OAc)2
DCM, RT
3a-f
+ AcOH
 
 
Scheme 2. Synthesis of thiopseudourea-based palladium(II) complexes 
 Abstract 
 iv 
CHAPTER-II: Thiopseudourea-Palladium(II) Complexes Catalyzed 
Suzuki and Hiyama Cross-Coupling Reactions of Aryl Halides 
This chapter describes the synthesis of biaryls from Suzuki and Hiyama 
cross-coupling reactions using thiopseudourea-Pd complexes as 
catalysts. A brief description has been given below. 
The palladium catalyzed Suzuki and Hiyama cross-coupling 
reactions are the “power tools of contemporary organic synthesis”. It 
consist the arylation of aryl halides, and has found application in natural 
products synthesis, materials science and bioorganic chemistry. The 
synthesis of biaryls from chloroarenes is of immense importance, since 
the C–Cl bond activation contributes to the fundamental understanding 
of the reactivity of such very stable bonds and they are cheaper and more 
widely available than their bromide or iodide counterparts. In particular, 
the activation of aryl chlorides has become an interesting task for Suzuki 
cross-coupling reaction. Therefore, nowadays there is much interest in 
the synthesis of catalysts that are able to activate aryl chlorides at lower 
catalyst loading. Attempts to activate the chloroarenes by strong basic 
ligands that provide the requisite electron density on palladium to 
undergo oxidative addition of Pd(0) facilitate the coupling reactions 
successfully. Phosphine containing ligands and palladacycles thus used 
to activate the chloroarenes are expensive, toxic and sensitive to air and 
temperature.  
 
 Abstract 
 v 
Furthermore, in compare to phosphine ligands, the N-donor 
ligands are stable, easily synthesizable and less toxic.  Consequently, it 
is highly desirable to develop phosphine-free Pd-catalysts for palladium 
catalyzed Suzuki reaction of aryl chlorides.  New homogeneous palladium 
catalysts based on the simple N-donor ligands were designed and 
developed for the Suzuki reaction of chloroarenes. The amide 
(deprotonated amide) ligands were chosen which is expected not only to 
stabilize the catalytically active palladium particles but also to provide 
the adequate electron density to the Pd(0) species to facilitate oxidative 
addition of chloroarenes. In this chapter, the synthesis of biaryls from 
Suzuki and Hiyama reactions using thiopseudourea-Pd(II) complexes as 
catalysts for the of aryl halides was described (Scheme 3 and 4). 
 
X
+ (HO)2B
Catalyst 3a
K2CO3
Solvent
R
RR1 R1
X = Br, Cl  
 
Scheme 3. Suzuki reaction of aryl halides catalyzed by palladium 
complex 3e. 
 
 
 
 
 
 
 Abstract 
 vi 
 
Br
+ (MeO)3Si
Catalyst 3a
LiOH.H2O
Ethyleneglycol
R
RR1 R1  
 
Scheme 4. Hiyama reaction of aryl bromides catalyzed by palladium 
complex 3e. 
The noticeable merits of these catalysts are:  
1) Compared to many of the earlier phosphine-free catalysts, these 
catalysts are air and thermally stable enough and active for the 
Suzuki reaction of activated aryl chlorides. 
2) In case of Hiyama reaction of aryl bromides, additional co-catalysts 
such as [NBu4]Br are required to achieve high catalytic activity. 
 
CHAPTER-III: Phosphine-free Thiopseudourea Ligated Pd(II) 
Complexes Catalyzed Heck and Sonogashira Cross-Coupling 
Reactions of Aryl Halides 
This chapter describes the thiopseudourea ligated Pd(II) complexes 
catalyzed Heck and Sonogashira cross-coupling reactions of aryl halides. 
A brief description has been given below. 
The palladium catalyzed C−C bond formation reactions such as the 
Heck and Sonogashira cross couplings reactions have found applications 
in the synthesis of natural products, pharmaceuticals and materials 
science. The great importance of C−C bond-forming reactions has 
 Abstract 
 vii 
encouraged the chemical community to search for highly active and 
stable palladium catalysts, which should be versatile and efficient. A 
number of phosphine-free ligands with different donor-functionalities 
such as N-heterocyclic, carbocyclic and anionic carbocyclic carbenes, 
oxazolines, schiff bases, pyridines, amines, imidazoles, pyrazoles, 
hydrazones, selenides, ureas and thioureas have been used for C−C 
cross coupling reactions. Most of these ligands are mono or bifunctional 
ligands. Therefore, it was assumed that the design and development of 
phosphine-free multifunctional ligands using different donor-
functionalities may provide stable and well-defined active palladium 
catalysts.  
 
X
[ X = I, Br, Cl]
R
Catalyst 3a
K3PO4
R1
CO2
nBu
R1
R
CO2
nBu
R
Catalyst 3a(0.001%)
LiOH.H2O
DMF, 130 oC
 
 
Scheme 5. Thiopseudourea-Pd(II) complex catalyzed Heck reactions of 
iodo-, bromo- and chloroarenes. 
 
 Abstract 
 viii 
In this chapter, the successful synthesis of stilbenes, aryl 
cinnamates and diphenylacetylenes by using thiopseudourea Pd(II) 
complexes as catalysts for the Heck and Sonogashira reactions was 
described (Scheme 5 and 6).  
 
+ Ph
Cat. 3e (0.01 mol%)
LiOH.H2O
DMF, 100 oC
Ph
R
X
R
X = I, Br  
 
Scheme 6. Thiopseudourea-Pd(II) complex catalyzed Sonogashira 
reactions of iodo and bromoarenes. 
 
The noticeable merits of these catalysts in C-C bond-forming reactions 
are:  
1) Compared to many of the previous phosphine-free catalysts, these    
    catalysts are air and thermally stable and have long lifetimes. 
2) Sonogashira reaction with iodo- and bromoarenes can be performed 
    at 100 ºC.  
3) Unlike most previously reported systems for C−C cross coupling 
reactions, additional additives such as [NBu4]Br or[PPh4]Cl are not 
required to achieve high catalytic activity. 
4) Catalysts have high turnover numbers with different aryl iodides 
and aryl bromides in Heck reactions. 
 
 Abstract 
 ix 
CHAPTER-IV: Application of Thiopseudourea ligated Pd(II) 
Complex as an Efficient Phosphine-free Catalyst for the Larock 
Indolization 
This chapter describes the application of thiopseudourea ligated 
Pd(II) complexes for Larock indolization and used as high turnover 
catalysts for the heteroannulation reaction of 2-iodoanilines. A brief 
description has been given below. 
The heteroannulation reaction has become not only one of the 
most exciting organic transformations for C–C and C–N bonds formation 
but also a benchmark to estimate the efficiency of a catalytic system. 
This powerful reaction has received considerable attention due to its 
functional group tolerance for synthesis of 2,3-disubstituted indoles and 
its application to a broad range of endeavors, ranging from synthetic 
organic chemistry to materials science. Therefore, several goals have to 
be achieved for its industrial application such as the use of inexpensive 
starting materials, achievement of high TON’s with less reactive aryl 
bromides and aryl chlorides, and the use of stable and inexpensive 
ligands. In this context, the design of new ligands and their Pd-catalysts 
that can affect the heteroannulation reaction of less reactive 2-bromo 
and 2-chloroanilines with both high activity and high efficiency has been 
a current important topic of research.  
 
 Abstract 
 x 
(1:1)x = I, Br, Cl
N
O
Pd
N
S
N
OAc
Catalyst.1
X
NHR
ph
N
R
ph
Catalyst 1
LiOH.H2O, DMF
 130 ºC
+ +
N
R
ph
R2
R2R1
R2
R1R1
 
Scheme 7. Heteroannulation reactions of 2-haloanilines and N-tosyl-2-
haloanilines catalyzed by thiopseudourea-Pd(II) complex. 
 
As the choice of ligand plays a major role in the efficiency of the 
catalyst, the phosphine ligands are the first choice to catalyze the 
heteroannulation reactions more actively and efficiently. However, the 
phosphine ligands are toxic, air-sensitive or quite expensive. Therefore, 
the development of phosphine-free Pd-catalysts has become another 
equally important topic of research. In this chapter, the synthesis of 2,3-
disubstituted indoles by thiopseudourea palladium(II) complexes bearing 
N-acylthiopseudourea/pyridyl ligands as N,N,O-donor coordinating sites, 
together with its performance as high turnover catalysts for the Larock 
indolization reactions of 2-haloanilines was described (Scheme 7). 
  
 Abstract 
 xi 
The thiopseudourea ligated Pd(II) complexes find their superiority 
over most of the phosphine-free catalysts with at least one of the 
following advantages: facile synthesis, thermal stability and structural 
versatility, easy handling, catalytic performance in air without any 
additives, achievement of high TON’s and presence of N-
acylthiopseudourea (RCONHCSNR1) hydrogen atom and the TON’s with 
2-iodoanilines and with hindered 2-bromoanilines, and activation of less 
reactive 2-chloroanilines. 
Significant Achievements: 
1) Development of phosphine-free palladium catalysts using N-donor 
ligands for the Larock indolization reactions 
2) The achievement of high turnover numbers for 2-iodoanilines with 
different diphenyl acetylenes 
3) Development of homogeneous phosphine-free palladium catalysts for 
the Larock indolization of less reactive 2-chloroanilines  
4) Application of Thiopseudourea palladium complexes as catalysts for 
heteroannulation reactions will open a new opportunity for the 
development of transition metal catalysis under phosphine-free 
conditions  


English

NEW APPROACHES FOR THE SYNTHESIS OF BIO-ACTIVE PIPERIDINE ALKALOIDS

Nimker, Charu

Novel Pyrrolo[2,1-c][1,4]benzodiazepine--Glycoside Prodrugs for Selective Therapy of Cancer and Conformationally Restricted PBD Dimers as
DNA Binding Anticancer Agents



SYNOPSIS
OF THE THESIS TO BE SUBMITTED FOR THE DEGREE OF 
DOCTOR OF PHILOSOPHY
IN CHEMISTRY

TO

Sri Venkateswara University
Tirupati







By
Venkatesh Tekumalla








Department of Chemistry
S. V. University College of Mathematical and Physical Sciences
Sri Venkateswara University
Tirupati-517 502
Dec-2007


Synopsis

The work in the thesis entitled “Novel Pyrrolo[2,1-c][1,4]benzodiazepine--Glycoside Prodrugs for Selective Therapy of Cancer and Conformationally Restricted PBD Dimers as  DNA Binding Anticancer Agents” has been undertaken with an aim to synthesize novel PBD molecules, for targeted delivery of the active moiety at the tumour site selectively and PBD dimers with enhanced DNA binding efficiency. The work carried out has been described in four chapters.

Chapter I: This chapter gives general introduction to cancer and its chemotherapy, covalent interactions of drug-DNA, particularly of pyrrolo[2,1-c] [1,4]benzodiazepine (PBD) antitumour antibiotics. It also gives an introduction to prodrugs and their classification and the objectives of present work.

Chapter II: This chapter deals with general introduction to antibody directed enzyme prodrug therapy (ADEPT), gene directed enzyme prodrug therapy (GDEPT) and prodrug mono therapy (PMT) strategies for selective therapy of cancer and recent developments in the area of prodrugs for selective therapy of cancer, followed by the description of design, synthesis and biological evaluation of PBD-galactoside prodrugs for targeted delivery of the active molecule at the tumour site with a potential for selective therapy of solid tumours, particularly the hepatocellular carcinoma, by ADEPT, GDEPT and PMT strategies. 

Chapter III: This chapter illustrates the importance of the enzyme -glucuronidase in selective treatment of solid tumours and also describes the synthesis and biological evaluation of PBD-glucuronide prodrugs for enhanced tumour selectivity by PMT, ADEPT and GDEPT strategies. 

Chapter IV: This chapter describes the design, synthesis and DNA binding affinities of novel conformationally restricted 1,4-cis and trans pyrrolobenzodiazepine dimers that are envisioned to possess improved DNA binding affinity. 
Chapter- I
General introduction to cancer, pyrrolobenzodiazepines as anticancer agents and prodrugs

Cancer is one of the dreadful diseases, which remained as a challenge to both physicians as well as scientists. Every year more than 20% of the population is affected by cancer and the rate of its induction throughout the world is increasing every year, making it an area of major focus for researchers. Cancer is not a single disease but a broad group, characterized by uncontrolled proliferation and spread of aberrant cells from their site of origin. At the simplest level, cancer cells may be regarded as having lost touch with the rest of the environment of the body so that they are no longer responsive to the controlling signals and interactions that occur continuously in normal healthy tissues. Cancer cells have some important properties that underlie the nature of the agent that causes the disease.
Treatment of cancer primarily consists of surgery, radiation and chemotherapy. The most common rationale for the use of chemotherapy is to control the cell population by cell-kill mechanism. Current chemotherapy consists of cytotoxic (cell-killing) agents and anti-hormonal drugs, which reduce the proliferative drive of the tumour. Many compounds with good cell-killing activity have been discovered, but few have found clinical utility. The lack of discrimination between a malignant (tumour) and normal tissue is one of the major hurdles. The cell-cycle dependency of many cytotoxic drugs and their frequent susceptibility to induce drug resistance and significant side effects are the difficulties often encountered during chemotherapy. The cell division in malignant cells occurs at a much faster rate in comparison to the normal cells and thus provides a potential target area for the development of chemotherapeutic agents. The role of chemotherapy is now apparent, in the effective treatment of a majority of solid tumours, as an adjuvant to initial therapy by surgical or radiotherapeutic procedures. Chemotherapy becomes critical for effective treatment for the reason that, only systemic therapy can cure micro metastases. The chemotherapeutic agents can be categorized based on their mode of action, like alkylating agents, antimetabolites, antibiotics and antimitotics. The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) belonging to the class of DNA-interactive antitumour antibiotics have the potential as regulators of gene expression with possible therapeutic application in the treatment of genetic disorders including cancer. The first PBD antitumour antibiotic anthramycin has been described by Leimgruber et. al. in 1963, and since then a number of compounds have been developed based on PBD ring system leading to DNA binding ligands. 


Figure 1

Pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a family of potent naturally occurring low molecular weight antitumour antibiotics originally isolated from various Streptomyces species. A number of naturally occurring and synthetic compounds based on PBD ring system, such as anthramycin, tomaymycin, DC-81 (Figure-1) and its dimers (presently SJG-136 is under clinical evaluation), have shown varying degrees of DNA binding affinity and anti-cancer activity. Their common interaction with DNA has been extensively investigated and it is considered unique since they bind within the minor groove of DNA forming a covalent aminal bond between the C11-position of the central B-ring and the N2 amino group of guanine base (Figure-2). 


Figure 2

Chapter- II
Design, synthesis and biological evaluation of PBD-β-galactoside prodrugs for potential application in selective chemotherapy of solid tumours by ADEPT, GDEPT and PMT strategies

Chemists have long sought the “magic bullet” for the treatment of cancer, a compound that would selectively kill cancerous tissues without affecting healthy cells. Perhaps a more realistic objective is selective delivery of a cytotoxic agent to the cancer cells, in other words, to maximize the concentration of the cytotoxic agent at the tumour while minimizing its concentration around healthy tissues.
A major focus of the current drug research is to improve drug targeting to internal target sites such as to solid tumours or specific organs. The objective of drug targeting, especially for cancer chemotherapy, is to enhance the effectiveness of the drug by concentrating it at the target site and minimizing its effects at non-target sites. 
The main pharmacophore i.e.; the imine functionality of PBD is modified into a carbinolamine prodrug, with an enzyme substrate coupled to the moiety at N10 position by a self-immolative spacer (Figure 3). In the present study prodrugs having the general structure pyrrolobenzodiazepine-spacer-β-galactoside have been designed, synthesized for application in selective chemotherapy of cancer. The prodrugs were evaluated for activation by the enzyme β-galactosidase. In the prodrug 1 and 2, the PBDs were connected to the β-D-galactoside moiety by a self-immolative spacer (Figure 3). 


Figure 3

The prodrugs are envisioned to enhance the tumour selectivity by ADEPT, GDEPT and PMT strategies.  Masking the imine functionality in PBD molecules by a sugar moiety also improved their water solubility. The chemical coupling and characterization of PBD-galactoside prodrugs, for selective delivery of the active principle to solid tumours especially to hepatocellular carcinoma, has been accomplished. The enzymatic activation of the prodrugs was employed for superior selectivity of the drugs to prevent tumour cell proliferation (Scheme 1).

Scheme 1

The delivery of PBD parent imines to the tumour site was expected to be enzyme specific i.e., the prodrug is activated to deliver the drug in the tissue, only in the presence of the enzyme β-galactosidase, leading to toxic effect. Two different cell lines, A375 and HepG2 were selected to probe the activation ability of the prodrugs by ADEPT and PMT strategies respectively. Therefore effective delivery of the drugs to the cells in the presence of externally administered E. Coli β-galactosidase was studied on human melanoma cell line A375.  The cells of Hep G2 are known to produce the enzyme β-galactosidase endogenously, and it was intended to exploit it, for selective therapy of hepatocellular carcinoma. The results were found accordingly, and the prodrugs were found to induce inhibition of cell division by the endogenous β-galactoside in HepG2 cells. The HepG2 cells were found to have toxic effect, by the activation of the prodrugs with the endogenous enzyme. These prodrugs have been found to be good substrates for use in PMT, ADEPT and GDEPT of solid tumours. The biological evaluation was made by CFSE antiproliferation assay.
In conclusion PBD--galactoside prodrugs with a potential for selective therapy of cancer by enzyme based therapies have been developed. 



Chapter- III
Design, synthesis and biological evaluation of PBD-β-glucuronide prodrugs for potential application in selective chemotherapy of solid tumours by ADEPT and PMT strategies

The histochemical studies of the cancerous tissues have shown the presence of β-glucuronidase in considerably high quantities compared to the normal tissues. Cancer tissues of breast are also reported to possess high quantities of the enzyme endogenously, which could serve the purpose of activating the prodrugs selectively in the breast tumours selectively. 
In continuation of our efforts to develop prodrugs of PBD anticancer agents for application in selective therapy of cancer, prodrugs having the general structure pyrrolobenzodiazepine-spacer-β-glucuronide have been designed and synthesized for activation by the enzyme β-glucuronidase. PBD-glucuronide prodrugs were conceptualized and synthesized because of the hydrophilicity of this functional group, the ability of glucuronide prodrugs to be preferentially activated at tumour cells targeted with β-glucuronidase antibody conjugates (ADEPT), and the strong antitumour activity observed after combined treatment with glucuronide prodrugs and β-glucuronidase-antibody conjugates or fusion proteins in animal models. In addition, glucuronide prodrugs have potential for cancer prodrug monotherapy (PMT) of tumours that over express or accumulate lysosomal β-glucuronidase.
As discussed in the previous chapter the imine functionality is modified as carbinolamine and masked with a self-immolative spacer connected to a -glucuronide moiety. DC-81 benzyl ether and an imine-amide dimer reported from our lab have been modified as glucuronide prodrugs 3 and 4 respectively (Figure 4).

Figure 4

The prodrugs were evaluated, for activation by the enzyme β-glucuronidase (Scheme 2) and their ability to kill the cancer cells selectively in the presence of the enzyme β-glucuronidase, and were found to be suitable for use in selective therapy of cancer by different enzyme prodrug strategies.


Scheme 2

In conclusion PBD--glucuronide prodrugs with a potential for selective therapy of cancer have been developed.
Chapter- IV
Design, synthesis and DNA binding efficacy of novel conformationally restricted 1,4-cis/trans pyrrolo[2,1-c][1,4]benzodiazpine dimers

Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers are synthetic sequence-selective interstrand DNA minor-groove cross-linking agents developed from the monomeric anthramycin family of naturally occurring antitumour agents. One possible explanation for the biological activity of PBD dimers is that they bind sequence-selectively to critical genomic regions containing an over-abundance of relevant binding sites, thereby blocking transcription of key genes essential for tumour growth and/or inhibiting DNA replication. This concept has been used to explain the activity of other sequence-selective DNA-binding agents. An alternative explanation is that, compared to normal cells, tumour cells have an inherently reduced ability to repair such stable and complex interstrand cross-linked adducts across the entire genome and so enter an apoptotic pathway. 
A series of 1,4-cis and trans dimers have been designed, synthesized and evaluated for their DNA binding affinity (Figure 5). The new isomers have been found to possess enhanced DNA binding affinity compared to their conformationally free saturated dimer.



Figure 5

In conclusion two new PBD dimers bridged by unsaturated, conformationally restricted chains have been designed synthesized and evaluated for DNA binding affinity, and were found to possess enhanced binding affinity compared to a saturated and conformationally free carbon chain bridged dimers. 
In conclusion the thesis describes the development of pyrrolo[2,1-c][1,4]benzodiazepine prodrugs for selective therapy of cancer and novel PBD dimers, as DNA interactive anticancer agents.



PAGE  









Prolyl hydroxylase inhibition and hypoxia inducible factor-1 as potential targets for inducing molecular adaptations in skeletal muscles: role of EDHB in improving physical performance.

Bhunia, Sanjoy Kumar

STRUCTURAL CHARACTERIZATION AND BIOLOGICAL INVESTIGATION OF MUSHROOM POLYSACCHARIDES

KUSAMPUDI, SHILPA

v 
 
ABSTRACT 
 The study attempted to mimic the state of obesity in vitro using 
3T3-L1 adipocytes, since they represent white adipose tissue which is 
responsible for obesity and related disorders. 3T3-L1 adipocytes were 
exposed to various conditions occurring during obesity in vivo such as (a) 
hyperglycemia (using high glucose), (b) hypoxia (using cobalt chloride 
(CoCl2)) and (c) macrophage infiltration (using RAW 264.7  conditioned 
medium (RCM). The study also attempted to assess the effect of a potential 
anti-adipogenic phytochemical (chlorogenic acid (CGA) - isolated from 
Cichorium intybus) and plant extract (Gymnema sylvestre methanol extract 
(GSM)) for their activity on the above conditions mimicking obesity.   
 A state of 25 mM glucose has been known to induce insulin 
resistance and hypertrophy in 3T3-L1 cells. To probe the role of glucose 
during hyperglycemia in adipocytes, the effect of various concentrations of 
glucose (45 mM, 65 mM, 85 mM and 105 mM glucose) were analyzed on 
3T3-L1 adipocytes. Differentiation of preadipocytes in the presence of 105 
mM glucose (high glucose) exhibited a significant decrease in adipogenesis 
through inhibition of CCAAT/enhancer-binding proteins (C/EBP ), sterol 
regulatory element binding protein 1c (SREBP1c) and peroxisome 
proliferator-activated receptor (PPAR ). Cells in the presence of high glucose 
(105 mM) showed increased cell proliferation through phosphotidyl inositol 3 
kinase (PI3K) / protein kinase B (Akt) dependent pathway. Furthermore, 
inhibition of p27 validated the induction of hyperplasia of activated 
preadipocytes under the state of high glucose. Nitric oxide and reactive 
oxygen species (ROS) production was induced at this condition leading to an 
increase in the expression of toll-like receptor 4 (TLR4), tumor necrosis factor 
vi 
 
 (TNF ) and nuclear factor kappa-light-chain-enhancer of activated B cells 
(NF B), thereby decreasing adiponectin production. This condition also 
showed insulin resistance through decrease in insulin receptor  (IR ), 
whereas an increase in glucose uptake was observed through activation of 
glucose transporter 1 (GLUT1). To summarize, 3T3-L1 adipocytes treated 
with 105 mM glucose mimicked the state of hyperplasia during obesity. 
 In this study, hypoxia condition was mimicked by treating 
differentiated adipocytes with 150 µM CoCl2 for 6, 12 and 24 h wherein, 
CoCl2 when treated for 12 h showed maximum expression of HIF1 . In 
comparison to control, cells under the state of hypoxia were found to inhibit 
lipid accumulation via C/EBP , leading to a decrease in adiponectin levels. 
Although, cytotoxicity was observed in cells treated with CoCl2, the 
expression of ROS was reduced in response to metabolic alteration during 
hypoxia. However, the levels of stress targets such as phosphorylated c-Jun 
N-terminal kinases (pJNK) and NF B were found to increase. Also, CoCl2 
treatment showed no significant change in GLUT1 levels however, glycogen 
synthesis was found to increase showing signs of hypertrophy. To 
recapitulate, 3T3-L1 adipocytes treated with 150 µM CoCl2 exhibited 
conditions imitating hypoxia during obesity. 
 To mimic the condition of macrophage infiltration in adipocytes 
during obesity, the effect of RCM on differentiated adipocytes was 
investigated for 6, 12 and 24 h. Maximum NO and ROS release along with an 
increase in inflammatory markers such as TNF , pJNK and phosphorylated 
extracellular regulated kinase (pERK1/2) was observed at 24 h of RCM 
treatment. Further, RCM treatment induced cytotoxicity in adipocytes and the 
increase in lipid content due to significant increase in lipofuscin content 
vii 
 
indicated the state of senescence.  This was validated by an increase in 
senescence markers such as p53, p21, retinoblastoma protein (pRb), p27 and 
cyclin D1. The cells treated with RCM showed an inhibition of insulin 
signalling, since expression of insulin receptor substrate 1(IRS1) and PI3K 
were found to decrease. The study clearly shows that RCM induced 
inflammation and senescence in adipocytes, thereby mimicking the state of 
macrophage infiltration induced senescence. 
 CDK5 is a protein kinase specifically expressed in the adipose 
tissue during obesity and is known to be activated by TNF  and various 
cytokines. Cells differentiated in the presence of 105 mM glucose and 
adipocytes treated with RCM for 24 h increased CDK5 levels significantly in 
comparison to control cells. But differentiated adipocytes when treated with 
150 µM CoCl2 for 12 h showed reduced levels of ROS and TNF  with no 
significant change in CDK5 levels, signifying the role of ROS and TNF  in 
the regulation of CDK5 expression. 
 As obesity is a complex disorder, it would be advisable to use 
treatments possessing multiple effects, thereby minimizing side effects. 
Bioactive molecules have an advantage of optimisation of the dosage for 
consumption. In contrast, the plant extracts are expected to show strong 
potential as activities are exhibited from a mixture of active molecules. Hence 
the study attempts to study the bioactivity exhibited by a CGA and GSM.    
 CGA (100 ng/ml) exhibited significant down regulation of PPAR , 
C/EBP , phosphorylated hormone sensitive lipase (pHSL) and CDK5 along 
with an increase in adiponectin level. CGA (100 ng/ml) showed effective 
regulation of targets involved during obesity, and hence was selected for 
understanding its role on the three in vitro conditions mimicking obesity. The 
viii 
 
cells differentiated with 105 mM glucose (hyperplasia model) when treated 
with CGA (100 ng/ml) showed significant decrease in GLUT1 expression and 
induced glycogen synthesis through increase in phosphorylated glycogen 
synthase kinase (pGSK3  - inactive form). CGA also inhibited CDK5 protein 
which was induced during hyperplasia. Treatment of CGA (100 ng/ml) in 
cells induced with hypoxia, showed an inhibition of HIF1 , pJNK and NF B 
leading to an increase in adiponectin levels. Adipocytes treated with RCM in 
the presence of CGA (100 ng/ml) reduced ROS production and lipofuscin 
levels along with decrease in p53, p21 and pRb levels, showing reversal of 
senescence. The increased expression of CDK5 in RCM treated adipocytes 
was also reduced during CGA treatment. 
 GSM (10 µg/ml) exhibited optimum bioactivity by inhibition of 
adipogenesis through down regulation of PPAR , SREBP1c and CDK5; 
however, an increase in C/EBP  and adiponectin was observed. GSM also 
inhibited the lipid protecting protein, perilipin with no significant change in 
pHSL levels. GSM was also investigated for its role on the three developed 
conditions mimicking obesity and results from the study clearly showed the 
potential of GSM in alleviating the various targets of obesity.  
 In conclusion, the present study has established in vitro models 
mimicking the conditions of obesity, characterized by hypertrophy/ 
hyperplasia, hypoxia and macrophage infiltration using 3T3-L1 adipocytes. 
The molecular programming involved during these conditions has also been 
elucidated through gene and protein expression studies. The therapeutic 
options for the developed conditions of obesity have been evaluated wherein 
CGA (100 ng/ml) and GSM (10 µg/ml) have been shown to be effective in 
alleviating various cellular targets involved during obesity. 


DEVELOPMENT AND ASSESSMENT OF IN VITRO CONDITIONS MIMICKING OBESITY AND THEIR ALLEVIATION USING NATURAL PRODUCTS

WAIKHOM, BIMOLATA

Title: Elucidating the Molecular Diversity Pattern of Major Bacterial Blight R-Genes and Identification of Candidate Bacterial Blight Resistance Genes in Rice
Abstract:
 In this thesis, we investigated the natural variation in genes which are involved in bacterial blight resistance. The aim of this study was to identify alternate source and form of resistance which will make elite susceptible rice varieties stand strongly against virulent Xoo strains. Phenotypic screening of 104 accessions including wild species, cultivars, landraces and introgression lines for resistance against five different Xoo strains DX011, DX127, DX020, DX015, and DX133 revealed that the wild accessions were highly resistant towards BB than the cultivated species. Through marker analysis two resistant forms of xa5 alleles were identified from O. sativa cultivar which may be useful for developing BB resistant lines. Polymorphisms of four BB resistance R genes Xa21, Xa26, Xa27 and xa5 were determined as quantified by average pair wise difference across all sites denoted by ‘π’.
Along the entire gene sequence, it was commonly observed that intron and non-coding region exhibited higher diversity for entire four locus. The allelic diversity was higher in O. nivara than O. sativa. In natural habitat there are more chances of intermixing genetic components in wild species by out crossing which increases the intensity of allelic diversity. Transition bias >1 was observed in all the genes which suggest that transition pattern of substitution is favoured over transversion during the course of evolution. During Xoo infection in wild rise O. nivara 81832 and O. longistaminata OL 1, out of 13 genes analysed, only seven genes were differentially expressed. These genes were constitutively expressed in both resistant and susceptible plants. OsBAKI, RPLD isoforms, EI10P8 and EI1K8 were found to induce and may be targeted for developing BB resistant lines. The proteome analysis was performed only in Xoo infected and water infiltrated resistant wild rice and only the up-regulated proteins were identified. Some of the protein identified in this study might play important roles in plant disease resistance, although further studies are required to verifiy the detailed localization and regulation of these proteins.


Elucidating the Molecular Diversity Pattern of Major Bacterial Blight R-Genes and Identification of Candidate Bacterial Blight Resistance Genes in Rice

CHANDA, CHANDRASEKHAR

Abstract

Thrombosis and related cardiovascular disorder are one of the major blood disorders of mankind.
Present day anticoagulant drugs to counter these disease conditions are expensive and give rise to
harmful side effects. Heparin and Warfarin are the two most widely used anticoagulants for the
treatment of thrombotic disorders like deep vein thrombosis, acute myocardial infarction,
thromboembolism and prevention of systemic embolism in patients with atrial fibrillation. One of the major limitations of these anticoagulants are their adverse side effects including hemorrhage, necrosis, osteoporosis and induced thrombocytopenia. Search for the newer class of anticoagulants with lower side effects and capable of digesting existing blood clots gained importance during the past two decades. Snake venom was found to be one of the richest natural sources for anticoagulant factors, mostly metalloproteinases. Some of the SVMPs are undergoing clinical trials worldwide for treating blood disorders. Ancrod / Viprinex is a defibrinogenating agent derived from the venom of the Malayan pit viper. Aggrostat puede from saw-scaled viper venom is presently used in combination with heparin and aspirin for management of unstable angina or myocardial infarction. It is also prescribed to patients who have undergone percutaneous trans-luminal coronary angioplasty to decrease the rate of refractory ischemic condition. But the search for ideal anticoagulant with minimal side effects has not ended.
In this study, we have purified two anticoagulant proteins form Indian monocled cobra venom
using various chromatographic techniques and named them as KT-6.9 and NKV 66.
KT-6.9 is a low molecular weight (6.9 kDa) polypeptide with strong antiplatelet properties. It has
sequence homology with several 3FTx family members. KT-6.9 specifically inhibited ADP,
thrombin and arachidonic acid induced platelet aggregation in a dose dependent manner which
emphasize the possibility of KT-6.9 binding to G protein coupled receptors (GPCRs). SPR analysisusing BIAcore X100 and other protein binding studies revealed the binding of KT-6.9 on theplatelet surface. Neurotoxicity studies on RPND models showed dose dependent and time
dependent inhibition of muscle contraction when treated with KT-6.9. It also inhibited
acetylcholine induced muscle contraction. KT-6.9 probably binding competitively with
acetylcholine on to post synaptic acetylcholine receptors on muscle cells.

NKV 66 is a 66 kDa metalloproteinase anticoagulant protein which showed chain specific
fibrinogenolytic activity where it has digested alpha chain of bovine fibrinogen in a dose and time dependent manner. NKV 66 is not a phospholipase and showed minimal hemolytic activity when tested on human RBC. NKV 66 completely hydrolyzed fibrin clots developed in vitro in 18 hours.Anticoagulant activity of NKV 66 was totally inhibited by EDTA treatment suggesting its
metalloproteinase activity whereas phenyl methyl sulfonyl fluoride treatment (PMSF), a serine
protease inhibitor did not affect its anticoagulant activity. NKV 66 was found highly thermostable and even survived exposure to 100º C for 1 min. Cleaving di-sulphide bonds by β-mercaptoethanol caused complete loss of activity and unfolding the protein with 4M urea also caused NKV 66 to loose of activity. NKV 66 inhibited ADP and collagen induced platelet aggregation process in a dose dependent manner possibly by enzymatic degradation of platelet receptors or by digesting fibrinogen and making it unavailable for platelet aggregation. NKV 66 was tested for disintegrin like activity on A549 cell lines. NKV 66 at concentration of (100μg) clearly affected the morphology of A549 cells. Most cells assumed a rounded appearance and detached from the basement membrane. To further assess the disintegrin like activity of NKV 66 cell adhesion assay was performed using collagen treated plates. About forty percent reduction in the cell adhesion was observed in case of NKV 66 treated cells. The results indicate disintegrins like activity of NKV 66 on A549 cells.


Studies on Anticoagulant Toxins from Indian Monocled Cobra Venom

G., Narmadha

Abstract 
 
 
ABSTRACT 
Lysozymes are hydrolases that are divided into 6 different families, based on their 
distribution and function. They are c-type, g-type, i-type, plant, phage and bacterial. C-type 
lysozymes are predominantly expressed among a wide variety of species. Most recently, 
lysozyme like (LYZL) proteins that belong to the c-type lysozyme family were identified, 
which include the six members namely LYZL1, SLLP1/LYZL3, LYZL4, LYZL5, LYZL6 
and LYZL7. Recently, the importance of mouse SLLP1 as a receptor for glycoprotein 
interaction during fertilization was reported. However, the exact role of LYZL proteins in 
general physiology and in male reproductive function in particular has not been studied. 
Hence, we attempted to characterize the rat LYZL proteins with specific reference to male 
reproductive tract function.  
 
Our results indicate that the rat LYZL proteins are encoded by separate genes and are not 
isoforms. Phylogenetic analysis showed that they are highly conserved and are distributed in 
various organisms. In silico analysis of the primary, secondary and tertiary structures of 
LYZL proteins revealed that they are homologous to chicken lysozyme, suggesting that Lyzl 
genes would have arose from a common ancestor. Among the LYZL proteins characterized, 
only LYZL1 and LYZL6 have conserved the lysozyme active site residues. Lyzl mRNA 
transcripts seem to be expressed predominantly in male reproductive tract. Their expression 
was not developmentally regulated and androgen independent. Immunostaining revealed the 
presence of these proteins in male reproductive tract tissues and on spermatozoa. LYZL1 and 
6 display peptidoglycan binding property and they possess muramidase, isopeptidase and 
antibacterial activities, whereas the remaining proteins did not. The role of LYZL proteins in 
sperm function was evaluated by incubating spermatozoa with LYZL6 antibodies. In vitro 
LYZL6 neutralization seems to inhibit calcium influx and acrosome reaction suggesting that 
Abstract 
 
 
they have important role in sperm function. In vivo studies using rats immunized with LYZL6 
displayed spermatozoa with reduced hypermotility and decreased fertility. Taken together, 
these results suggests that LYZL6 may have a role in sperm function. 


Elucidation of the Functional Role of Rat Lysozyme-Like Proteins in the Male Reproductive Tract

HANSI, PRISCILLA D.

Hypoglycemic and Insulin Sensitizing Effect of Naringenin on High Fat Diet Fed, Streptozotocin Induced Type 2 Diabetic Rats

Thakur, Shilpa

Abstract 2014 
 
 1 
 
Scope: Intestine is known to play a central role in folate homeostasis as it is the 
main route of absorption of folates in mammalian cells. Folates, being negatively 
charged molecules cannot pass through the cell membranes and therefore 
requires specific membrane transporters for its transport in and out of the cell. 
Reduced folate carrier (RFC) and proton coupled folate transporter (PCFT) are 
the specific folate transporters present on the membrane surfaces of the intestinal 
cells. Besides these two, folate receptor (FOLR) is another transporter which is 
not expressed in intestine under normal conditions but its expression is observed 
in colon in cancer conditions. Folate being an essential micronutrient is needed 
for normal human health and well being. Normal microflora of large intestine 
can synthesize folate however the amount is insufficient to fulfill the metabolic 
requirements of the body. Therefore, exogenous supply of this vitamin either 
through diet or through nutritional supplements is important. Deficiency of this 
vitamin has been commonly seen in developing as well as in well developed 
countries. Although numerous factors contribute in development of folate 
deficiency but inadequate dietary folate intake and chronic alcohol intake are 
among the most frequent causes. So in the present study, we sought to examine 
the regulatory mechanisms underlying the effects of folate deficiency and 
ethanol exposure on intestinal folate transport and to check the reversibility of 
such effects. 
Methods: Caco-2 cells (in vitro) were grown under two different set of 
conditions. In the first set, cells were grown for 5 generations in control and 
folate deficient medium. Following treatment, one subgroup of cells was shifted 
on folate sufficient medium and grown for 3 more generations. In another set of 
experiments, cells were grown in the medium containing 100mM ethanol for 96 
hr. Thereafter, one subgroup of cells was shifted on ethanol free medium and 
grown for next 72hr. For in vivo studies, rats were fed on FD diet for 3 and 5 
months and after 3 months of FD treatment, one group of rats was shifted on 
Abstract 2014 
 
 2 
 
normal folate containing diet. For ethanol treatment, rats were given 1g 
ethanol/kg body weight/day either for 3 or 5 months and after 3 months of 
ethanol treatment, one group of rats was left for 2 months without ethanol. 
Results: Dietary folate deficiency as well as ethanol exposure resulted in a 
significant decrease in folate levels within Caco-2 cells, rat intestinal tissue as 
well as in rat serum and thus, reflected the folate deficient conditions after both 
the treatments.  The folate transport process in cell line as well as in rats revealed 
an increased uptake of folic acid across both membrane surfaces i.e. BBM and 
BLM in case of folate deficiency. On the other hand, a decrease in folic acid 
uptake was observed in case of ethanol treatment in both Caco-2 cells as well as 
in rats. Transport was found to be specific with the help of structural analogs of 
folic acid and inhibitors of folate transporters. The alterations in folate transport 
characteristics were associated with alterations in maximal velocity both in case 
of folate deficiency and ethanol treatment; however, a decrease in affinity of 
folate transporters was also observed in ethanol exposure. The increase/decrease 
transport of folate in folate deficiency/ethanol exposure was associated with a 
parallel increase/decrease in the mRNA as well as protein expression of folate 
transporters respectively in both Caco-2 cells as well as in rats. However, when 
cells and rats were shifted to control conditions after treatment, transport as well 
as expression of these genes restored to the control level. Folate deficiency and 
ethanol exposure were found to have no impact on promoter methylation of 
PCFT and RFC in both Caco-2 cells and rats. As far as mRNA stability of 
transporters is concerned, a decrease in mRNA half lives of PCFT and RFC was 
observed on folate deficiency treatment however ethanol exposure resulted in an 
increase in mRNA half lives of PCFT and RFC and hence suggesting some 
adaptive response. Neither treatment had any effect on mRNA half life of FOLR. 
Results of nuclear run on assay have revealed an increase in rate of synthesis of 
folate transporters on folate deficiency, however, a decrease in rate of synthesis 
Abstract 2014 
 
 3 
 
was observed on ethanol exposure in Caco-2 cells. These results were in a 
positive correlation with the data on expression of transporters during both the 
treatments. The expression analysis of various transcription factors involved in 
the regulation of PCFT, RFC and FOLR, revealed significant changes in their 
mRNA levels. Further studies with ChIP experiment revealed a decreased 
binding of SP1 transcription factor to the promoter region of all three folate 
transporters on ethanol treatment however folate deficiency resulted an 
increased binding of SP1 to the promoter region of RFC and PCFT only. 
Conclusion: Overall, increased folate uptake and expression of its transporters 
under folate deficiency conditions can be attributed to enhanced rate of 
transcription of the folate transporters and increased binding of SP1 to the RFC 
and PCFT promoters in Caco-2 cells. A reduced binding of SP1 to the promoter 
region of all the three folate transporters appears to be the contributing 
regulatory mechanism behind decreased expression and reduced rate of 
synthesis of folate transporters upon ethanol exposure of intestinal cells. 


Regulation of intestinal folate transport upon ethanol exposure and folate deficiency

Jivrajani, Mehul Navinchandra

 Page XIV 
 
Targeted Delivery of shRNA to Cancer Cell 
Abstract 
Novel targeted delivery for shRNA was developed by exploiting bacterial minicells. In this 
work, shRNA against VEGF A was packaged in minicells. These minicells were linked with 
folic acid for active targeting of tumor cells via folate receptor in vitro and in vivo. In order to 
achieve this, E.coli PB114 was selected as a minicell producing strain. Subsequently, E.coli 
PB114 was transformed with pEZ43G-D in order to confirm segregation of plasmid DNA 
into the minicells. Minicells were purified from E.coli PB114 pEZ43G-D by a combination of 
ceftriaxone lysis and filtration. Ultrapure minicells have been obtained by this new method. 
Total number of minicells was found to be 3.81 × 1010 minicells/ml from 200 ml of starting 
culture. Cancer cell lines, LNCaP, HeLa and KB have be n selected as positive control 
whereas A549 was selected as negative control in terms of folate receptor overexpression. 
shRNA expression vectors, psNIPERDH1A1 and psNIPERDU6A2, were transfected in 
selected cell lines and gene expression of VEGF A was analyzed by reverse transcriptase PCR 
in order to validate gene silencing of VEGF A. Both the expression vectors were found to 
downregulate VEGF A mRNA but psNIPERDU6A2 silenced VEGF A more efficiently, 
hence psNIPERDU6A2 was selected for in vitro and in vivo delivery. In vitro delivery was 
studied by delivering 109 minicells from each the group, 1) FAminicellspSUPERneoScramble 2) 
minicellspsNIPERDU6A2, 3) 
FAminicellspsNIPERDU6A2 in selected cell lines and expression of VEGF 
A was analysed by RT-PCR.  Expression of VEGF A did not changed in any of the group in 
A549 cell line. In contrast, expression of VEGF A reduced significantly in 
FAminicellspsNIPERDU6A2 treated group when compared with other two groups in positive 
control cell lines. Moreover, uptake of folic acid conjugated minicells was found to be 
through receptor mediated endocytosis. Tumor xenograft of A549, LNCaP and KB cells were 
developed in immunosuppressed C57 BL6 mice. Animals were divided in four groups and 
treated with 1) Saline, and 109 of respective minicells 2) FAminicellspSUPERneoScramble 3) 
minicellspsNIPERDU6A2, 4)
 FAminicellsPSNIPERDU6A2 intravenously. There was a gradual increase 
in the tumor volume till the end of treatment in all four groups of A549 xenograft. Whereas in 
case of LNCaP and KB xenograft, there was a significant decrease in tumor volume in 
FAminicellspsNIPERDU6A2 treated group as compared to other groups. Similarly, expression of 
VEGF A was found to be same in all the groups when compared with the saline treated group 
 Page XV 
 
in A549 xenograft. On the contrary, significant downregulation of VEGF A was found in 
FAminicellsPSNIPERDU6A2 treated LNCaP and KB xenograft. Finally, In vivo biodistribution 
study of FITC loaded FAminicellspsNIPERDU6A2 revealed majority of fluorescence localize in the 
tumor followed by liver and heart. These results suggest that majority of 
FAminicellspsNIPERDU6A2 were distributed in tumor tissue which can be attributed to both 
passive and active targeting of FAminicellspsNIPERDU6A2. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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